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Hyperfibrinogenemia and sphingolipid-mediated cerebrovascular permeability and memory impairment during TBI

TBI期间高纤维蛋白原血症和鞘脂介导的脑血管通透性和记忆障碍

基本信息

批准号：
10855710
负责人：
DAVID LOMINADZE
金额：
$ 75万
依托单位：
UNIVERSITY OF SOUTH FLORIDA
依托单位国家：
美国
项目类别：
财政年份：
2023
资助国家：
美国
起止时间：
2023-08-15 至 2024-07-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/10855710
关键词：
Address Alzheimer&apos s Disease Alzheimer&apos s disease related dementia Alzheimer&apos s disease risk Astrocytes Attenuated Avidity Binding Blood Blood Proteins Blood Vessels Brain Caveolae Complex Confocal Microscopy Cortical Contusions Crossbreeding Data Dementia Deposition Development Disease Electron Microscopy Electrons Endothelial Cells Endothelium Enzymes Exocytosis Extravasation Fibrinogen Fluorescence Fluorescence Resonance Energy Transfer Fumonisins Generations Glucosylceramides Goals Heart Diseases Immunohistochemistry Impaired cognition In Situ In Vitro Inflammation Injury Intercellular adhesion molecule 1 Knockout Mice MAPK3 gene Measures Mediating Memory Memory Loss Memory impairment Methods Microscopy Mitochondria Modeling Morbidity - disease rate Mus N-palmitoylsphingosine Pathogenicity Pathway interactions Peripheral Nervous System Permeability Phosphorylation Pilot Projects PrP Production Proteins Research Research Priority Resistance Role Short-Term Memory Signal Transduction Small Interfering RNA Sphingolipids Sphingomyelins Testing Tracer Transgenic Organisms Traumatic Brain Injury United States National Institutes of Health Vascular Cognitive Impairment Vascular Diseases Vascular Endothelial Cell blood-brain barrier permeabilization brain endothelial cell caveolin 1 cerebrovascular dihydroceramide desaturase in vivo inhibitor insight knockout gene loss of function mortality mouse model neuroinflammation new therapeutic target novel object recognition protein complex receptor serine palmitoyltransferase thermozymocidin transcytosis

项目摘要

Project Summary Increased blood content of fibrinogen (Fg), e.g. hyperfibrinogenemia (HFg) is a risk factor for Alzheimer's disease (AD). As it occurs during neuroinflammation, it is a risk factor for AD & related disorders (ADRD), including vascular cognitive impairment & dementia (VCID). Our goal is to characterize the role of a novel mechanistic pathway, namely Fg-sphingolipid-caveolae nexus during traumatic brain injury (TBI) as an example of ADRD, which is accompanied with HFg. Our data indicate that cortical contusion injury (CCI)-induced HFg enhances cerebrovascular permeability mainly via caveolar protein transcytosis leading to Fg deposition in extravascular space and resulting in greater formation of Fg and cellular prion protein complexes resulting in short-term memory (STM) reduction, typically occurring in AD. We showed that HFg increased Fg binding to endothelial intercellular adhesion molecule-1 (ICAM-1) enhancing formation of functional caveolae & exocytosis via activation of ERK-1/2 and caveolin-1 (Cav-1). We found that HFg upregulated de novo sphingolipid synthesis pathway & caveolar transcytosis using mitochondrial ATP. A selective inhibitor of sphingolipid synthesis pathway ameliorated the HFg- triggered caveolar protein transcytosis in mouse brain endothelial cells, & importantly, reduced cerebrovascular protein transcytosis after CCI. Based on these results, we hypothesize that at elevated levels, Fg, through binding to its endothelial receptor ICAM-1, activates sphingolipid production resulting in increased caveolar protein transcytosis in ECs that is supported by ATP generated in mitochondria. A corollary hypothesis is that an enhanced caveolar Fg transcytosis contributes to STM reduction similar to that during AD. Specific aims are: (1) Determine whether the increased interaction of Fg with endothelial ICAM-1 enhances formation of functional caveolae via production of Cer, GlcCer, and SPM sphingolipids and thereby increases caveolae-mediated protein transcytosis. (2) Determine whether increased interaction of Fg with ECs enhances mitochondrial activity using up energy for caveolar protein transcytosis. (3) Determine whether inhibition of de novo sphingolipid synthesis attenuates HFg- induced increased formation of functional caveolae decreasing caveolar protein transcytosis and thus, ameliorates the STM reduction during TBI. To test mechanisms of HFg-induced caveolar transcytosis via sphingolipid signaling we will use WT, HFg, and endothelium-specific serine palmitoyltransferase long chain-2 gene knockout (Sptlc2endo-/-) and HFg/Sptlc2endo-/- mice generated by Cre/lox method, with or without CCI. The dual-tracer probing method will be used to define changes in caveolar transcytosis. Loss-of-function strategy (specific siRNAs against ICAM-1 & Cav-1) and specific inhibitors of sphingolipid synthesis will be used. Immunohistochemistry and intravital, confocal, electron, total internal reflection fluorescence, and fluorescence resonance energy transfer microscopies will be used. Changes in STM will be assessed by a novel object recognition and Y-maze tests. These studies will provide insight into the role of sphingolipids in mechanisms of cerebrovascular permeability & may unveil new therapeutic targets for one of the leading causes of VCID.

项目摘要血液中纤维蛋白原（Fg）含量增加，例如高纤维蛋白原血症（HFg）是阿尔茨海默病的危险因素（AD）。由于它发生在神经炎症期间，它是AD及相关疾病（ADRD）的风险因素，包括血管性认知障碍和痴呆（VCID）。我们的目标是描述一种新的机械途径，即创伤性脑损伤（TBI）期间的Fg-鞘脂-小窝关系，作为ADRD的一个例子，它伴随着HFg。我们的数据表明，皮质挫伤（CCI）诱导的HFg增强脑血管通透性主要通过小窝蛋白转胞吞作用导致血管外Fg沉积空间，并导致更多的Fg和细胞朊病毒蛋白复合物的形成，从而导致短期记忆 (STM)减少，通常发生在AD。我们发现HFg增加了Fg与内皮细胞间的结合粘附分子-1（ICAM-1）通过激活ERK-1/2促进功能性小窝和胞吐作用的形成和Caveolin-1（Cav-1）。我们发现HFg上调了鞘脂的从头合成途径&小窝使用线粒体ATP进行胞吞转运。鞘脂合成途径的选择性抑制剂改善了HFg- 触发小鼠脑内皮细胞中的小窝蛋白转胞吞作用，重要的是， CCI后的蛋白质转胞吞作用。基于这些结果，我们假设在升高的水平下，Fg通过结合与其内皮受体ICAM-1结合，激活鞘脂产生，导致小窝蛋白增加在EC中的转胞吞作用由线粒体中产生的ATP支持。一个必然的假设是，小窝Fg转胞吞作用有助于STM减少，类似于AD期间。具体目标是：（1）确定 Fg与内皮细胞ICAM-1相互作用的增加是否通过 Cer、GlcCer和SPM鞘脂的产生，从而增加小窝介导的蛋白质转胞吞作用。 (2)确定增加的Fg与EC的相互作用是否增强线粒体活性，小窝蛋白转胞吞作用(3)确定从头鞘脂合成的抑制是否减弱HFg- 诱导功能性小窝形成增加，减少小窝蛋白转胞吞作用，从而改善 TBI期间STM减少。检测HFg通过鞘脂诱导小窝转胞吞的机制我们将使用WT、HFg和内皮特异性丝氨酸棕榈酰转移酶长链-2基因敲除（Sptlc 2 endo-/-）和HFg/Sptlc 2 endo-/-小鼠。双示踪剂探测方法将用于定义小窝转胞吞的变化。功能丧失策略（针对 ICAM-1和Cav-1）和鞘脂合成的特异性抑制剂。免疫组织化学和活体，共聚焦、电子、全内反射荧光和荧光共振能量转移显微术将用于STM的变化将通过新的物体识别和Y-迷宫测试来评估。这些研究将提供对鞘脂在脑血管通透性机制中的作用的深入了解，并可能揭示新的 VCID主要病因之一的治疗靶点。